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Published Ahead of Print on March 11, 2016, as doi:10.3324/haematol.2015.141465.
Copyright 2016 Ferrata Storti Foundation.
Bio-engineered and native red blood cells from cord blood exhibit
the same metabolomic profile
by Dhouha Darghouth, Marie-Catherine Giarratana, Lydie Oliveira, Séverine Jolly,
Tiffany Marie, Samia Boudah, Nathalie Mario, Christophe Junot, Luc Douay,
and Paul-Henri Romeo
Haematologica 2016 [Epub ahead of print]
Citation: Darghouth D, Giarratana MC, Oliveira L, Jolly S, Marie T, Boudah S, Mario N, Junot C,
Douay L, and Romeo PH. Bio-engineered and native red blood cells from cord blood exhibit the same
metabolomic profile. Haematologica. 2016; 101:xxx
doi:10.3324/haematol.2015.141465
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Bio-engineered and native red blood cells from cord blood exhibit the same
metabolomic profile
Dhouha Darghouth1* Marie-Catherine Giarratana2 Lydie Oliveira,3,6 Séverine Jolly,7
Tiffany Marie,7 Samia Boudah,8 Nathalie Mario,9 Christophe Junot,10 Luc Douay,2,7,11
and Paul-Henri Romeo3,6
1
Sorbonne Universités, UPMC Univ Paris 06, Inovarion, Inserm UMRS 938, Centre
de Recherche Saint-Antoine, Paris, France.
2
Sorbonne Universités, UPMC Univ Paris 06, Inserm UMRS 938, Centre de
Recherche Saint-Antoine, Paris, France.
3
CEA/DSV/iRCM/LRTS, 92265 Fontenay-aux-Roses cedex, France.
4
Inserm U967, 92265 Fontenay-aux-Roses cedex, France.
5
Université Paris-Diderot, Paris 7, France.
6
Université Paris-Sud, Paris 11, France.
7
Sorbonne Universités, UPMC Univ Paris 06, EFS, Inserm UMRS 938, Centre de
Recherche Saint-Antoine, Paris, France.
8
Commissariat à l'Energie Atomique et aux Energies Alternatives/Direction des
Sciences du Vivant/Institut de Biologie et de Technologie de Saclay/Service de
Pharmacologie et d'Immunoanalyse/Laboratoire d'Etude du Métabolisme des
Médicaments, CEA-Saclay, 91191 Gif-Sur-Yvette, France; GlaxoSmithKline - Centre
de recherche F.Hyafil, Villebon-sur-Yvette, France.
9
AP-HP, Hôpital Saint-Antoine, Service de Biochimie A, F-75012 Paris, France
10
Commissariat à l'Energie Atomique et aux Energies Alternatives/Direction des
Sciences du Vivant/Institut de Biologie et de Technologie de Saclay/Service de
Pharmacologie et d'Immunoanalyse/Laboratoire d'Etude du Métabolisme des
Médicaments
11
Sorbonne Universités, UPMC Univ Paris 06, APHP, Inserm UMRS 938, Centre de
Recherche Saint-Antoine, Paris, France.
*
Corresponding author : Dhouha Darghouth : [email protected]
1
The increasing need for red blood cells (RBCs) together with the lack of donors have
made the in vitro production of RBCs a major medical challenge.1 We have recently
developed a method to produce mature RBCs in vitro, starting from bone marrow,
peripheral blood, leukapheresis or cord blood-derived CD34+ cells. This method is
based on a three-steps protocol using a serum-free medium supplemented with
cytokines.2 Here, we assess the quality of the RBCs produced in vitro using
metabolomics, developed recently for RBC analysis3 or reticulocytes.4 We show that
native reticulocytes (nRets) and culture-derived reticulocytes (cRets) exhibit very
similar metabolomics signatures, 80% of their metabolites being expressed at similar
levels. The homology is almost complete (90% of metabolites expressed at similar
levels) when are compared RBCs produced from nRets and from cRets. Furthermore,
the differentially expressed metabolites are not involved in relevant functions of
RBCs. Altogether, these results show that RBCs originating from in vitro produced
reticulocytes phenocopy native RBCs, a great promise to their successful use in
blood transfusion.
Human cord blood CD34+ cells were differentiated into erythroid progenitors that
were then grown on a murine MS-5 stromal cell line which provides a
microenvironment allowing terminal erythroid differentiation including enucleation
(Supplementary Figure 1).2 At day 15 the majority of the cells produced were
reticulocytes as assessed by thiazole orange staining (89%±10%) and by expression
of the transferrin receptor (89%±10 CD71+). The Mean Cell Volume (MCV), Mean
Corpuscular Hemoglobin Concentration (MCHC) and Mean Cell Hemoglobin (MCH)
of these cells were within the ranges we have been observing in the laboratory: 128
±11.4 fL (130–138 fL), 22± 0.2g/dL (18–25 g/dL) and 29 ±2.8 pg (23–35 pg),
respectively. 2,5
As culture conditions may lead to metabolic stresses that could alter RBC
functionality, we first compared the metabolomes of cRets and nRets. nRets were
purified from cord blood by supermagnetic microbead selection using a CD71
antibody6 and were more than 96% ± 2% (n=5) pure. Reticulocytes metabolites were
analyzed using two liquid chromatographic (LC) systems coupled to high resolution
mass spectrometry operated in both negative and positive modes of electrospray
ionization.3,7 The wide chemical diversity of metabolites is best assessed by this
2
optimal metabolomic approach that can detect highly polar metabolites such as sugar
derivatives or amino acids using hydrophilic interaction LC, as well as fatty acids or
acylcarnitines using reverse phase chromatography. Eighty-six metabolites of
biological relevance were identified (Supplementary Table 1) and analyzed in nRets
and cRets. We found that the concentration of metabolites related to glycolysis or
glutathione metabolism, two important pathways of RBCs functions were similar in
the two types of reticulocytes (Figure 1-2). Twenty-one metabolites displayed at least
a two fold increase or decrease in concentration in cRets compared to nRets (Figure
1). Among the metabolites with increased concentration in cRets, six out of ten were
amino acids (glutamine, alanine, serine, ornithine, threonine and asparagine) (Figure
1). The increased concentration of these amino acids in the cRets is likely to be due
to their high concentration in the culture media used and to the presence of their
transporters at the surface of reticulocytes.8 Metabolites related to membrane
integrity (carnitine, O-propanoyl-carnitine, acetyl-carnitine and R-butyryl-carnitine)
were all derived from carnitine and had a decreased concentration in cRets (Figure
1-2). RBCs from sickle cell patients9 and from patients with overhydrated
stomatocytosis10 also have increased concentrations of carnitine and acetyl-carnitine.
These increased concentrations are associated with high membrane turnover and
repair mechanisms as carnitine and acetyl-carnitine are used as reservoir of
activated acyl group for the turnover and the repair of RBCs membrane.11 Thus, our
results may indicate that in vitro differentiated reticulocytes exhibit a lower membrane
turnover and repair than circulating reticulocytes, possibly because membrane stress
may be lower in static conditions. We also found a decreased concentration of
malate, 2-oxoglutarate and succinate that are part of the Krebs cycle that takes place
in mitochondria that are still present in young reticulocytes.12 These decreased
concentrations might be the consequence of a high glucose concentration in the
culture media, driving efficient ATP synthesis through glycolysis rather than the
Krebs cycle in cRets compared to nRets. Finally, the two metabolites that have the
most decreased cRets concentration are ergothioneine and stachydrine (proline
betaine) that share the same transporter13 and that are not present (ergothioneine) or
present at very low levels (stachydrine) in culture medium. Ergothioneine is not
synthetized by human cells but by actinobacteriae or filamentous fungi.14 Its presence
in nRets from cord blood is the likely consequence of ergothioneine intake by the
mother and transfer to nRets where ergothioneine fuels a very efficient anti-oxidative
3
pathway.15
nRets and cRets were further differentiated into mature nRBCs and cRBCs2 by
extending co-culture further for 8 days. All parameters assessed in cRets established
that the maturation process had clearly started at day 23 (Supplementary Table 2) as
evidenced by the decrease in nucleic acids and mitochondria content, by lower
CD71/CD36 (thrombospondin receptor) expression and to a lesser extent, by smaller
MCV and increased MCH. Lastly, as expected, the membrane rigidity diminished
during the cRets maturation (Supplementary Figure 2). A similar maturation pattern
was demonstrated for nRets exhibiting a decrease in CD71 expression and nucleic
acids and mitochondria contents (Supplementary Figure 3).
When the metabolomes of nRBCs and cRBCs were compared, most metabolites
with increased cRets levels had returned to nRBCs levels in cRBCs (Figure 3). This
result was due to an increased concentration of the differential metabolites in nRBCs
and was consistent with the culture medium origin of the increased concentration of
these metabolites in cRets. As for the metabolites with decreased concentrations in
cRets, only the carnitine and its metabolite derivatives and stachydrine/ergothioneine
had a comparative decrease in concentration in cRBCs (Figure 3). These results
strengthen the exogenous contribution of stachydrine/ergothioneine in nRBCs and
indicate that in vitro produced cRBCs might have decreased membrane turnover and
repair, possibly due to the less stressing conditions of cell culture, compared with
blood circulation. An alternative hypothesis to account for the differences in
metabolites is that they originate from inter-individual variations, since native
reticulocytes and CD34+ cells from which cRets were differentiated, were purified
from separate cord bloods. However covariance analysis established that for 7 out of
the 11 metabolites covariance was below the level of significance (25-30%
,Supplementary Table 3), thus arguing against inter-individual differences. The
differences of the remaining 4 metabolites may originate from inter-individual
variations, or from technical issues. Thus out of 86 metabolites, upon differentiation
only 7 seem to vary due to culture conditions, and 4 to sample origin, confirming that
cRBCs nearly phenocopy nRBCs.
We have previously established the proof of principle for transfusion of in vitro
generated reticulocytes and developed procedures to validate these bio-engineered
cells. Here, using a metabolomics approach, we provide evidence that reticulocytes
either native or produced in vitro exhibit similar metabolomic signatures. Almost
4
identical metabolomic signatures were obtained after maturation into cRBCs. This
result strengthens the robustness of the protocol we have developed to produce
reticulocytes to be used for transfusion, an alternative to classical transfusion
products, in the hope to circumvent donor scarcity in such cases as patients with very
rare blood groups.
Acknowledgments
We thank Jean-Philippe Rosa for his help in writing and critically editing this
manuscript, Alexandre Seye (Prophilomic) for his assistance in data analyses, Pierre
Buffet for kindly putting his laboratory premises and the LORCA at our disposal.
5
References
1.
Douay L, Andreu G. Ex vivo production of human red blood cells from
hematopoietic stem cells: what is the future in transfusion? Transfus Med Rev.
2007;21(2):91-100.
2.
Giarratana MC, Kobari L, Lapillonne H, et al. Ex vivo generation of fully mature
human red blood cells from hematopoietic stem cells. Nat Biotechnol. 2005;23(1):6974.
3.
Darghouth D, Koehl B, Junot C, Romeo PH. Metabolomic analysis of normal
and sickle cell erythrocytes. Transfus Clin Biol. 2010;17(3):148-150.
4.
Malleret B, Xu F, Mohandas N, et al. Significant biochemical, biophysical and
metabolic diversity in circulating human cord blood reticulocytes. PLoS One.
2013;8(10):e76062.
5.
Giarratana MC, Rouard H, Dumont A, et al. Proof of principle for transfusion of
in vitro-generated red blood cells. Blood. 2011;118(19):5071-5079.
6.
Brun A, Gaudernack G, Sandberg S. A new method for isolation of
reticulocytes: positive selection of human reticulocytes by immunomagnetic
separation. Blood. 1990;76(11):2397-2403.
7.
Boudah S, Olivier MF, Aros-Calt S, et al. Annotation of the human serum
metabolome by coupling three liquid chromatography methods to high-resolution
mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2014;966:3447.
8.
Benderoff S, Blostein R, Johnstone RM. Changes in amino acid transport
during red cell maturation. Membr Biochem. 1978;1(1-2):89-106.
9.
Darghouth D, Koehl B, Madalinski G, et al. Pathophysiology of sickle cell
disease is mirrored by the red blood cell metabolome. Blood. 2011;117(6):e57-66.
10.
Darghouth D, Koehl B, Heilier JF, et al. Alterations of red blood cell
metabolome in overhydrated hereditary stomatocytosis. Haematologica.
2011;96(12):1861-1865.
11.
Arduini A, Mancinelli G, Radatti GL, et al. Role of carnitine and carnitine
palmitoyltransferase as integral components of the pathway for membrane
phospholipid fatty acid turnover in intact human erythrocytes. J Biol Chem.
1992;267(18):12673-1281.
12.
Ney PA. Normal and disordered reticulocyte maturation. Curr Opin Hematol.
2011;18(3):152-157.
13.
Grundemann D, Harlfinger S, Golz S, et al. Discovery of the ergothioneine
transporter. Proc Natl Acad Sci U S A. 2005;102(14):5256-5261.
14.
Genghof DS, Vandamme O. Biosynthesis of Ergothioneine and Hercynine by
Mycobacteria. J Bacteriol. 1964;87:852-862.
15.
Chaudiere J, Ferrari-Iliou R. Intracellular antioxidants: from chemical to
biochemical mechanisms. Food Chem Toxicol. 1999;37(9-10):949-962.
6
Figure legends
Figure 1: Metabolic signatures of cRets and nRets are similar.
Metabolomes of cRets from blood from 4 independent umbilical cords and nRets
from 5 independent umbilical cords were analyzed by LC/MS followed by
identification in the metabolite database.5 A diagram of high- and low-expressed
metabolites is shown. cRets data are expressed as log2 fold relative to the average
expression level in nRets for each metabolite. Metabolites expression was
considered significantly different between cRets and nRets when log2R was above
+2 or below -2 (black bars) and was considered identical when log2R was between
+2 and -2 (gray bars). Statistical significance was assessed with a Mann-Whitney
test: ns, not significant; *p ≤ 0.05.Ф This value has been arbitrarily set at 0 (-∞ as a
log20) for ease of display.
Figure 2: Clustering of metabolites expressed in cRets and nRets
A. Shown are metabolites in equal (blue) or different (red) amounts in cRets or
nRets. B. Expanded view of metabolites expressed at different levels in cRets and
nRets, and ranked according to characteristics or function. C. Expanded view of
metabolites expressed at similar levels in cRets and nRets, and ranked according to
characteristics or function.
Figure 3: Metabolic signatures of red blood cells obtained by maturation of
cRets and nRets.
A. Diagram of high- and low-expression metabolites in cRBCs compared to nRBCs.
Black bars correspond to log2R for metabolites expressed at different levels in cRets
and nRets. Gray bars correspond to log2R for metabolites expressed at different
levels in cRBCs and nRBCs. Statistical significance was assessed by Mann-Whitney
test: NS, not significant; * p ≤ 0.05; Ф This value has been arbitrarily set at 0 (-∞ as a
log20) for ease of display. B. Metabolites differentially expressed in cRBCs and
nRBCs were clustered according to function (membrane integrity or stability) or
characteristic (amino acids). The number of metabolites per cluster is indicated.
7
Supplemental Methods
1-1 Biological material and sample preparation
Cultured red blood cells
Umbilical Cord Blood (CB) from normal full-term deliveries were obtained with
informed consent. Cord Blood CD34+ cells were isolated by supermagnetic
microbead selection using Mini-MACS columns (Miltenyi Biotech, Bergisch Glodbach,
Germany) (94 ± 3 % purity). The cells were cultured in erythroid differentiation
medium (EDM) based on IMDM (Iscove modified Dulbecco’s medium, Biochrom,
Germany) supplemented with stabilized glutamine, 330 µg/mL human holo-transferrin
(BBI solutions, Sittingbourne, UK), 10 µg/mL recombinant human insulin (FEF,
Denmark), 2 IU/mL heparin Choay (Sanofi, France) and 5% solvent/detergent virus
inactivated (S/D) plasma (Etablissement Français du Sang, France).
The procedure comprised three steps. In the first step (day 0 to day 8), 104/mL
CD34+ cells were cultured in EDM in the presence of 100 ng/mL SCF (PeproTech,
Neuilly-sur-Seine, France), 5 ng/mL IL-3 (PeproTech) and 3 IU/mL Epo (Eprex,
Janssen-Cilag, Issy-les-Moulineaux, France). On day 4, one volume of cell culture
was diluted in four volumes of fresh medium containing SCF, IL-3, Epo. In the second
step (day 8 to day 11), the cells were resuspended at 105/mL and seeded onto MS-5
at 2x104cells/cm2 in EDM supplemented with Epo. In the third step (day 11 to day
15), the non adherent cells were recovered, centrifuged, diluted in fresh medium not
containing cytokines (the volume of culture medium was doubled at day 11 as
compared to day 8). Cells were re-seeded onto stromal cells. The cultures were
maintained at 37°C in 5% CO2 in air.
Stromal cells
The murine MS-5 stromal cell line was expanded in αMEM medium containing
ribonucleosides, deoxyribonucleosides, Glutamax (Life Technology) and 10% fetal
calf serum (FCS). At confluence, adherent cells were collected after treatment of the
cultures for 7-10 min with trypsin-EDTA 1X (Life Technology) at 37°C. The recovered
cells (usually 106/25cm2) were washed and replated at 4000/cm2 in αMEM medium
supplemented with Glutamax and 10% FCS. Cultures were incubated at 37°C under
5% CO2 and adherence was usually reached after one week.
Deleukocytation
The cell suspensions were purified by passage through a deleukocyting filter to
eliminate the expelled nuclei and residual erythroblasts. The purity of the cRBC
samples was 99.3 ± 0.2 % after filtration and the filtered suspensions were washed
twice in PBS.
Maturation of native and cultured reticulocytes
To induce the maturation of the RETc, cells were washed, resuspended at 57x106/ml in IMDM + 5% human AB plasma without cytokines and co-cultured on a
new stromal layer. The culture supernatants were renewed twice a week. Cultures
were maintained at 37°C under 5% CO2.
Reticulocyte separation
Native reticulocytes were isolated from peripheral blood by an immunomagnetic
method (Miltenyi Biotec). Briefly, the cells were incubated with anti-CD71 microbeads
1
(Miltenyi Biotec) and the labeled cells were enriched on Mini-MACS columns. The
reticulocyte content of the CD71-purified population was controlled by thiazole
orange staining.
Flow cytometric analyses
Cells were labeled with unconjugated or fluorescein isothiocyanate (FITC) or
phycoerythrin (PE)-conjugated antibodies. Anti-CD235 (glycophorin A) -PE, antiCD71-PE or –FITC, anti-CD36-FITC and anti-CD34-PE antibodies (Beckman
Coulter, Marseille, France) were used for phenotyping. Analyses were performed on
CYAN ™ ADP flow cytometer (Beckman Coulter) using Summit software.
Reticulocyte count
3x105 cells were washed in PBS (pH 7.4) and incubated with 300 µL of Retic-count
solution (Retic-count/Thiazole-Orange, BD Biosciences, Le Pont-de-Claix, France)
for 30 min at room temperature. A negative control was carried out by incubating the
cells with PBS alone.
Mitochondrial Membrane Potential
Resdietraethylbenzimidazolyl-carbocyanine iodide (JC-1) was used to measure the
mitochondrial membrane potential. When this cationic dye accumulates into the
mitochondrial membrane it forms aggregates leading to a shift in fluorescence from
green to orange. Briefly, 5x105 cells were washed and resuspended at 106/mL in
prewarmed EDM, and then incubated at 37°C under 5% CO2 for 15 min with 20
ng/mL JC-1 (Life Technologies) or without JC-1 (for negative control). Cells were
generously washed twice in prewarmed PBS and immediately analyzed on a CYAN
™ ADP flow cytometer (Beckman Coulter) using Summit software (Excitation
488nm; Emission 530-590 nm).
Deformability measurements
The cell flexibility of cRBC and native reticulocytes was determined using a laser
diffraction technique [LORCA (Laser-assisted Optical Rotational Cell Analyzer); R&R
Mechanotrics, Hoor, The Netherlands] as extensively described previously1 2The cell
deformability was expressed as elongation index (EI) which was recorded
continuously at various shear stresses in the range 0.3-30 Pa. The EI value at 30 Pa
was referred to as EImax.
Preparation of RBC lysates and metabolite extraction
109 packed native or cultured cells were pelleted and resuspended in qs 1ml Milli-Q
water, and boiled for 3 minutes. After centrifugation at 1430g for 3 minutes at 4°C,
the supernatant was ultrafiltered with centrifugal filter devices, molecular weight cutoff
of 30 000 followed by 10 000 (Amicon Ultra-4 centrifugal filter devices) at 4000g for
20 minutes at 20°C. The final ultrafiltrates were divided into 4 aliquots and frozen at 80°C until mass spectrometry analysis.
Before injection into the chromatographic system, the samples were dried under
nitrogen. Two aliquots of each sample were diluted in 105µL H2O /External
Standards mixture (ExS) in 105µL of H2O/ACN/ExS, 40/60/5 µl for HILIC analysis . A
quality control sample (QC) was obtained by pooling RBC samples subsequently
diluted ½, ¼ and ⅛. A QC sample was injected after each dilution series and after
each 10 samples to assess signal repeatability and stability.
2
1-2/ Instrumentation and LC-MS acquisitions:
LC-MS analyses were performed using a Dionex Ultimate chromatographic
system (Thermo Fisher Scientific, Courtaboeuf, France) coupled to an Exactive
spectrometer (Thermo Fisher Scientific, Courtaboeuf, France) fitted with an
electrospray source operated in the positive and negative ion modes. The software
interface was Xcalibur (version 2.1) (Thermo Fisher Scientific, Courtaboeuf, france).
The mass spectrometer was calibrated before each analysis in both ESI polarities
using the manufacturer’s predefined methods and recommended calibration mixture
provided by the manufacturer (external calibration).
The ultra-high performance liquid chromatographic (UHPLC) separation was
performed on a hypersil GOLD C18 1.9µm, 2.1mm x 150mm column at 30°C (Thermo
Fisher Scientific, les Ulis, France). Hydrophilic interaction liquid chromatographic
separation (HILIC) was achieved on a Sequant ZICpHILIC 5µm, 2.1 x 150mm at
15°C (Merck, Darmstadt, Germany). All chromatographic systems were equipped
with an on line prefilter (Thermo). Experimental settings for each LC/MS condition are
described below.
The Exactive mass spectrometer was operated with capillary voltage at -3 kV
in the negative ionization mode and 5 kV in the positive ionization and capillary
temperature at 280°C. The sheath gas pressure and the auxiliary gas pressure were
set, respectively, at 60 and 10 arbitrary units with nitrogen gas. The mass resolution
power of the analyzer was set to 50000 m/Δm, full width at half maximum (FWHM) at
200u, for singly charged ions. The detection was achieved from 50 to 1000 u in the
positive ionization mode, from 95 to 1000u for reverse phase (RP) chromatography in
the negative ionization mode and from 85 to 1000u for HILIC conditions.
Mobile phases for UHPLC were 100% water in A and 100% ACN in B, both
containing 0.1% formic acid. Regarding HILIC, phase A consisted of an aqueous
buffer of 10mM of ammonium carbonate in water with ammonium hydroxide to adjust
basicity to pH 10.5, whereas acetonitrile was used as solvent B. Chromatographic
elutions were achieved under gradient conditions as follows:
(i) RP based system: the flow rate was set at 500µL/min. The elution consisted of an
isocratic step of 2 minutes at 5% phase B, followed by a linear gradient from 5 to
100% of phase B for the next 11 minutes. These proportions were kept constant for
12.5 min before returning to 5% B for 4.5 min.
(ii) HILIC based system: the flow rate was 200µL/min. Elution started with an
isocratic step of 2 min at 80% B, followed by a linear gradient from 80 to 40% of
phase B from 2 to 12 min. The chromatographic system was then rinsed for 5 min at
0% B, and the run ended with an equilibration step of 15 min.
2-4/ Data processing:
All raw data were manually inspected using the Qualbrowser module of Xcalibur
version 2.1 (Thermo Fisher Scientific, Courtaboeuf, France). Automatic peak
detection and integration were performed using the XCMS software package.3
Grouping of features was performed using CAMERA software.4 Features were then
annotated by matching their accurate measured mass at ± 10 ppm with theoretical
ones contained in biochemical and metabolomic databases such as KEGG5 HMDB6,
Metlin7 by using an informatics tool developed in R language, and also by our
spectral database according to accurate measured masses and chromatographic
retention times.8, 9
3
Signal Drift Correction and Batch Effect Removal
Within each peak table, intensities were corrected for signal drift and batch effect by
fitting a locally quadratic (loess) regression model to the QC values.10,11 The α
parameter controlling the smoothing was set to 1 to avoid overfitting. Once the peak
tables were normalized, metabolites with a coefficient of variation (CV) of their QC
values >25% were filtered out.
4
Supplemental references
1.
Hardeman MR, Ince C. Clinical potential of in vitro measured red cell
deformability, a myth? Clin Hemorheol Microcirc. 1999;21(3-4):277-84.
2.
Giarratana MC, Rouard H, Dumont A, Kiger L, Safeukui I, Le Pennec PY, et al.
Proof of principle for transfusion of in vitro-generated red blood cells. Blood. 2011
Nov 10;118(19):5071-9.
3.
Smith CA, Want EJ, O'Maille G, Abagyan R, Siuzdak G. XCMS: processing
mass spectrometry data for metabolite profiling using nonlinear peak alignment,
matching, and identification. Anal Chem. 2006 Feb 1;78(3):779-87.
4.
Kuhl C, Tautenhahn R, Bottcher C, Larson TR, Neumann S. CAMERA: an
integrated strategy for compound spectra extraction and annotation of liquid
chromatography/mass spectrometry data sets. Anal Chem. 2012 Jan 3;84(1):283-9.
5.
Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes.
Nucleic Acids Res. 2000 Jan 1;28(1):27-30.
6.
Wishart DS, Tzur D, Knox C, Eisner R, Guo AC, Young N, et al. HMDB: the
Human Metabolome Database. Nucleic Acids Res. 2007 Jan;35(Database
issue):D521-6.
7.
Smith CA, O'Maille G, Want EJ, Qin C, Trauger SA, Brandon TR, et al.
METLIN: a metabolite mass spectral database. Ther Drug Monit. 2005
Dec;27(6):747-51.
8.
Boudah S, Olivier MF, Aros-Calt S, Oliveira L, Fenaille F, Tabet JC, et al.
Annotation of the human serum metabolome by coupling three liquid chromatography
methods to high-resolution mass spectrometry. J Chromatogr B Analyt Technol
Biomed Life Sci. 2014 Sep 1;966:34-47.
9.
Roux A, Xu Y, Heilier JF, Olivier MF, Ezan E, Tabet JC, et al. Annotation of the
human adult urinary metabolome and metabolite identification using ultra high
performance liquid chromatography coupled to a linear quadrupole ion trap-Orbitrap
mass spectrometer. Anal Chem. 2012 Aug 7;84(15):6429-37.
10.
van der Kloet FMB, I.; Verheij, E. R.; Jellema, R. H. Analytical Error Reduction
Using Single Point Calibration for Accurate
and Precise Metabolomic Phenotyping. J Proteome Res. 2009;8(11):5132−41.
11.
Dunn WB, Broadhurst D, Begley P, Zelena E, Francis-McIntyre S, Anderson
N, et al. Procedures for large-scale metabolic profiling of serum and plasma using
gas chromatography and liquid chromatography coupled to mass spectrometry. Nat
Protoc. 2011 Jul;6(7):1060-83.
5
Supplemental Figures
Supplementary Figure 1: In vitro production of RBCs from cord blood CD34+:
The method used comprises three steps. In the first step, CD34+ cells purified from
Umbilical Cord Blood are grown for 8 days in medium supplemented with stem cell
factor (SCF), interleukin-3 (IL-3) and erythropoietin (Epo). Then (second step), the
cells are cultured on the murine MS-5 stromal cell line in the presence of Epo (day 8
to day 11). In the third step, all exogenous factors are withdrawn and the cells are
grown further on MS-5 stromal cells without cytokines for up to 5 days. This protocol
permits massive erythroid expansion and complete differentiation into mature
cultured Red Blood Cells (cRBCs).
6
Supplementary Figure 2: Deformability profiles of cultured reticulocytes before
and after maturation:
Cultured reticulocytes (cRets) were recovered at day 15 of culture and purified using
a deleukocyting filter. Culturing the purified cRets for 8 supplementary days set up a
maturation step. The deformability (elongation index, EI) of the cRets (n=3) was
evaluated on LORCA over a range of shear stresses (0.3-30 Pa) before (black curve)
and after the maturation step (grey curve).
7
A
B
C
Supplementary Figure 3: Maturation of native reticulocytes into nRBCs :
After 8 days of maturation, native reticulocytes (nRets) exhibit a decrease in (A)
Transferrin receptor (CD71) expression assessed by flow cytometry, (B) nucleic acids
assessed by thiazole orange staining and (C) mitochondria assessed by JC-1
staining.
8
Supplemental Tables
Supplementary Table 1: List of ion observed in RBCs human samples, using
UHPLC/Exactive and ZICpHILIC/Exactive
9
* nd in C18
** nd in HILIC
(a) Identification based on accurate mass
(b) Identification based on C18 retention time
(c) Identification based on HILIC retention time
(d) Identification based on MS spectra
10
Supplementary Table 2: Immunophenotypic and functionality caracterisation
of cultured reticulocytes (cRets) and red blood cells (cRBCs)
Retic Count (%)
CD 71 (%)
GlycoA (%)
CD 36 (%)
VGM (fL)
TCMH (pg)
CCMH (g/dL)
cRets
89±10,4
89±10,4
100±0
37±34,8
128±11,4
29±2,8
22±0,24
cRBCs
8±1,6
26± 7
100±0
3±3,3
114±7,4
30±3,8
26±2,2
The results are expressed as the mean ± SEM
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Supplementary Table 3: Genetic correlation for the 11 metabolites differentially
expressed in native (nRBCs) and culured red blood cell (cRBCs) using
coefficient of variation (CV, %)
Stachydrine
cRBCs
nRBCs
14.6
9.3
R-butyrylcarnitine
95.9
32.9
Taurine
Carnitine
12.0
7.4
29.9
27.8
O-propanoylcarnitine
59.1
60.9
Trimethylamine
Oxide
103.2
84.5
Acetyl
1,3-Bis-P-DSorbitol DHA Hexose-P
carnitine
Glycerate
10.5
28.6
23.3
28.0
39.3
28.3
7.3
50.8
25.8
22.1
No correlation is associated with CV below 25-30%.
12